Effect of age on leukotriene B4 production in guinea pig whole blood
| Autor: | Penelope A. Pechous, Courtney L. Scaife, Stephen M. Spaethe, Eldwin L. VanAlstyne |
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| Rok vydání: | 1992 |
| Předmět: |
Male
medicine.medical_specialty Thromboxane Leukotriene B4 Guinea Pigs Enzyme-Linked Immunosorbent Assay Biochemistry Phospholipases A chemistry.chemical_compound Internal medicine medicine Animals Chromatography High Pressure Liquid Whole blood Pharmacology Leukotriene Arachidonic Acid biology Age Factors Blood Cell Count Phospholipases A2 Endocrinology chemistry Eicosanoid Prostaglandin-Endoperoxide Synthases biology.protein Arachidonic acid Cyclooxygenase Eicosanoid Production |
| Zdroj: | Biochemical pharmacology. 43(4) |
| ISSN: | 0006-2952 |
| Popis: | We evaluated the effect of age on eicosanoid production in guinea pig blood. Heparinized blood from 7–10-day, 6-week, or 6-month-old guinea pigs was incubated with 150 μM arachidonic acid (AA) for 5 min, followed by stimulation with A23187 (20 μg/mL) for an additional 10 min at 37°. The reaction was terminated by centrifugation, and the production of plasma leukotriene (LT) B 4 and C 4 , prostaglandin E 2 (PGE 2 ), and thromboxane B 2 (TXB 2 ) was determined by enzyme-linked immunoassay (ELISA). LTC 4 , PGE 2 , and TXB 2 formation were unaffected by age. In marked contrast, production of LTB 4 was increased 4- to 5-fold as age increased from 7–10 days (9.51 ± 2.07 ng/mL) or 6 weeks (8.83 ± 1.81 ng/mL) to 6 months (40.57 ± 9.66 ng/mL). To determine the effect of age on the total eicosanoid product profile, blood was stimulated in the presence of [ 14 C]AA, and plasma metabolites were separated by reverse-phase high pressure liquid chromatography (RP-HPLC) and quantitated using on-line radiochemical detection. In addition to increased LTB 4 production, a modest increase in 12-hydroxyeicosatetraenoic acid (12-HETE) production was also observed in the 6-month-old animals. Previous studies have demonstrated interference of 12-HETE in the immunoassay of LTB 4 . Therefore, to validate the authenticity of the plasma leukotriene ELISA measurements, samples were precipitated with methanol and fractionated by RP-HPLC. The fractions co-eluting with [ 3 H]LTB 4 or [ 3 H]LTC 4 were dried under vacuum and reconstituted in ELISA buffer, and leukotrienes were quantitated. As seen previously, following HPLC purification LTB 4 production remained significantly elevated in the 6-month-old guinea pigs, whereas LTC 4 production was unaffected by age. To further document the selectivity of this effect on LTB 4 production, we evaluated the effect of increasing age on cyclooxygenase or phospholipase A 2 (PLA 2 ) activity. Neither cyclooxygenase nor PLA 2 activity was elevated as animals matured. In conclusion, the capacity of whole blood to produce LTB 4 , but not LTC 4 , TXB 2 , or PGE 2 , was elevated markedly in older animals. |
| Databáze: | OpenAIRE |
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